Keywords: Biomonitoring; Drinking Water; Groundwater; stygobionta; Crustacean; Copper;
In order to improve water body quality, the European Commission enacted the Water Framework Directive [EC 2000/60/EC] which pledged that all European Union member states would work to achieve good qualitative and quantitative status of all water bodies including groundwater. The directive focuses on the recently revised directive for priority substances, as well as catchment-specific substances [Directive 2013/39/EU] , for which environmental quality standards [EQS] should not be exceeded [2008/105/EC].
Numerous studies reveal increasing levels of heavy metals and other pollutants in ground and surface water a problem that is expected to worsen in the future [1, 21, 46, 66, 68, 72]. For instance, copper concentration in soil and water has increased through anthropogenic discharge including; “smelting, mining, industrial, and domestic waste emission, and the application of fertilizers, sewage sludge, algicides, fungicides, and molluscicides” [11, 21, 63]. Usually, the concentration of copper in lakes and rivers can range from 0.5 to 1,000 ppb [with an average concentration of 10 ppb], whereas the average copper concentration in groundwater is typically around 5 ppb though it can be as high as 2,783 ppb . However, the concentration of copper and other pollutants typically fluctuate due to seasonal changes, water level, heavy rainfall or other irregular events [7, 61]. Additional risks to human health from polluted water can occur due to specific events like chemical spills such as the Sandoz accident at River Rhine in 1986 as well as deficient chemical filtering and bioterrorism .
The recommended World Health Organization [WHO] threshold for copper in drinking water is 2 mg/L , a guideline which has been implemented in countries like Germany and Australia. The US regulation is stricter, with the Environmental Protection Agency [EPA]’s “Lead and Copper Rule” [LCR] restricting average copper concentration to a maximum of 1.3 mg/L. To the best of our knowledge, the Federal-Provincial-Territorial Committee on Drinking Water [CDW], published by Health Canada, has defined the lowest threshold of 1 mg/L of any country.
These types of regulations require the appropriate devices for realization of the implemented limits. Regardless of whether drinking water originates from surface-, ground- or from reuse of sewage water, strict observance of mutually defined thresholds must be controlled to ensure its safety . For instance, in Germany, depending on turnover size, previous test results and type of contaminant, drinking water must be analysed 1-12 times a year . However, generally high levels and continuously fluctuating water body pollution demonstrate the need of strict and continuous controlled water quality to protect sensitive water organisms and ensure safe drinking water sources .
In recent decades biological early warning systems [BEWS] using living organisms ranging from bacteria and algae to daphnids, gammarids, and even fish have been developed and tested [4, 5, 19, 30, 33, 45, 76]. Cell-based biosensors rely on oxygen consumption [bacterial respirometers], nitrification, carbon dioxide production, bacterial growth, fluorescence, or photosynthesis [5, 14, 49]. In contrast, BEWS implementing animals are based on behavioural changes. High sensitivity towards a broad range of toxicants. Fast response rates of both Daphnidae and Gammaridae qualify them as good species for the use as live biomonitors [11, 13, 30, 33, 76].
Gammarus fossarum [Koch, 1835] [Crustacea, Amphipoda] is widely distributed in streams in the Northern hemisphere. As a decomposer, they mainly feed on fallen leaves including their biofilm. Due to high prevalence, G. fossarum is broadly used as a bioindicator for stream water quality control and plays a key role in the aquatic food web [15, 16, 57]. Gammarus has been shown to be sensitive to a variety of toxicants, responding with characteristic behavioural responses such as increased ventilation or decreased locomotion [9, 25, 26, 29, 30, 33, 50]. Gammaridae have been previously used in monitoring Rhine water and the U.S Environmental Protection Agency [EPA] has published 1996 guidelines for gammarid acute toxicity tests [OPPTS 850.1020] [28, 29, 75]. Gammarids have also demonstrated their reliability in numerous ecotoxicological tests . The high sensitivity to a variety of toxicants and ease of breeding, together with its abundant occurrence, qualifies G. fossarum as an ideal species for online monitoring.
The planktonic Daphnia magna [Crustacea, Phyllopoda] lives in fish free open waters and feeds mainly on algae and organic detritus . Therefore, they show a high basal activity. This r-strategist is able to reproduce asexually, resulting in predominantly female clones. If environmental conditions deteriorate, male offspring are produced to incorporate sexual reproduction, leading to diversification and adaptation . The ease of breeding and their utility in a variety of experimental scenarios made daphnids, especially D. magna and D. pulex, one of the most frequently used organisms for ecotoxicological experiments in aquatic systems . This has led to daphnids being used in commercially available online biomonitoring systems [13, 40]. However, the use of clones, depending on the genotype and previous conditions, may lead to differences in sensitivity towards toxicants [6, 67].
In this study, we introduce the groundwater species Niphargopsis casparyi [Pratz, 1866], the stygal relative of Gammaridae and Proasellus slavus [Remy, 1948] as a potential candidate species for online biomonitoring of ground- and drinking water using the Multispecies Freshwater Biomonitor [MFB] . Groundwater is characterized by almost surreal conditions, like exclusion of light, low food or oxygen availability . Therefore, stygobionta are characterized by a slower metabolism which has led to longer life cycles compared to their surface-water relatives . Niphargidae represent the stygal relative of Gammaridae and as some of the largest stygal organisms are predominantly predacious, but also feed on plant detritus or bacterial film. Proasellus slavus [Remy, 1948] [Isopoda, Asellota] feeds mainly on detritus. According to Bork et al. , the specie is a good example to illustrate adaptations to the groundwater environment. Asellus aquaticus, living in surface water, lives up to one year and lays eggs every month. In contrast, P. slavus can live up to 15 years and lays eggs only every 2-4 years . Despite its lower basal activity, which is the trait we are using as indication for wellbeing, low demands and longevity are of great advantage.
Stygal organisms are generally characterized by a lower basal activity, but due to their longevity and their lower energetic demands, they may represent good test subjects for online biomonitoring. Reduction in maintenance would make online biomonitoring more time and cost efficient. Additionally, we expect groundwater Amphipods to be more sensitive to toxic stress, as usually they are exposed to lower toxicant levels compared to surface waters.
Replicates of up to five individuals were placed in a beaker [250 ml] in a total volume of 200 ml solution. Replicates of more than five individuals were placed in 600 ml beakers in a volume of 400 ml. Beakers were sealed with foil and aerated for G. fossarum. Copper-solutions were freshly prepared and renewed weekly. G. fossarum was fed on a piece of an alder leaf [d 3 cm], with the loss providing a rough estimation of feeding behaviour at the end of the exposure. Stygal species fed on fine detritus which was sampled together with the animals. Mortality of all species was checked twice a week. Furthermore, activity of G.fossarum and N. casparyi was measured twice a week for 2 hours in the MFB to check for changes in activity patterns throughout the exposure time. Therefore, 2-3 individuals, randomly chosen from each of the replicates, were inserted individually in the test chambers in copper-free creek water using a plastic pipette.
Over the period of two months, using the MFB, locomotion and ventilation activity of Niphargopsis casparyi was continuously monitored. Test chambers were placed in a 20-l flow-through aquarium with a flow-through rate of ~3.6 l/min raw water, as to be seen in Figure 1. Trace length was determined as four minutes, followed by a pause of six minutes. Two types of behaviour were defined, covering movements between 0 and 5 Hz. Band 1 summarizes the impedance changes with frequencies of 0.5-2 Hz, representing locomotion. Band 2 summarizes higher frequencies from 2.5-5 Hz, which portray ventilation. Low capture rates for niphargids constrained us to implement only three individuals in December. In January, subject number could be increased to eight. The setup and survival of niphargids was checked weekly. Due to the low capture in December, only dead animals were replaced. In January, if any animals died, all were replaced.
Alarm settings for the stepwise increasing Cu-pulse proceeded with the following. Immobility signal was elicited if 25% of the channels did not show any activity [mortality alarm]. Changes in behaviour were calculated as deviations from the prediction based on the last 3 records [ARIMA model]. The software gave a behavioural alarm if the activity measurement deviated once for more than 10% or 5% from the mean of the last 3 recordings of Band 1 [locomotion] or Band 2 [ventilation], respectively.
Furthermore, alarm settings and data resolution in the MFBsoftware were changed. One measurement lasted four minutes followed by only two minutes pause [instead of 6 minutes before] in order to gain higher time resolution of the MFB-Biomonitor. Immobility alarm was elicited by 25% [instead of 50% before] of the individuals. The prediction was calculated using the last five measurements [instead of 3 recordings], in order to incorporate the variability of the individuals and thus reduce false positive alarms. Tolerated deviation from Band 1 [locomotion] and Band 2 [ventilation] were 10% and 3%, respectively.
SigmaPlot [Systat Software, San Jose, CA] was used to perform statistical analysis to test for significant behavioural differences between the replicates upon acute copper exposure. Since the replicates were unpaired and most data sets did not pass the normal distribution test [Shapiro-Wilk], the non-parametric oneway analysis of variance on ranks [Kruskal Wallis] with pairwise multiple comparisons [Tukey] was applied. Furthermore, LC50 for different endpoints was determined using GraphPad Prism Version 5.01. LC50 for G.fossarum and E.serrulatus could be defined by the “log[inhibitor] vs. response - Variable slope” analysis. Lower number of replicates and tested concentration levels restricted us to calculate LC50 for P.slavus using the “log[inhibitor] vs. response” function with a standard slope of -1. The 24-h LC50 of D.magna was determined with the Probit method using MS Excel Version 2016.
Used solutions were weekly analysed using high performance liquid chromatography [HPLC] at 280 nm by Technologiezentrum Wasser [TZW] in Karlsruhe.
Threshold (TWV) [mg/l]
Since all four field monitoring experiments showed similar and reproducible results, two are presented exemplary in the following. Figure 6 depicts the averaged locomotion [black] and ventilation [green] of niphargids as well as the Daphnia Biomonitor toxicity index [BD TI] [dark red]. Over the first 13 days exposure to raw water intake from lake Constance, niphargids’ locomotive activity was on average of 13% ± 7% SD [n= 3]. High variability was due to one individual which only occasionally showed activity right from the beginning of the experiment. On day three niphargids started and continued to show increased ventilation, a behaviour rarely seen in N. casparyi, that normally indicates stress, which in turn was affirmed with a reaction of the BD TI. A comparison to the physicochemical data revealed a single peak in pH and a parallel drop in SAK, during this period. pH increased within two hours from 7.96 to 8.91 and dropped in the following hour back to 7.9. SAK decreased retrograde to 0.01 and rose back to 1.97. Simultaneous to the increase in pH, niphargids started ventilating. Although stress ventilation activity decreased after two days, niphargids kept ventilating occasionally, indicating sustained and undefined water contamination or a long-lasting impact. The same was true for the daphnids until they were replaced on the 7th day. Afterwards the BD presented basal values. Throughout the remaining six days, fluctuation in locomotive activity appeared to increase, indicating an avoidance response. On the last day an increase in BD TI coincided with increasing ventilation and fluctuating locomotion in the niphargids. This second suspicious coincidence could not be attributed to physicochemical parameters.
The next day, 22 hours after exposure started, water inflow was enabled and marked with a blue vertical line. With an inflow of ~3.6 l/min the concentration halves every 5.5 minutes. Assuming no contamination of the raw water, concentration in the aquarium was below 10 μg/l within 37 minutes. During the two days recovery period only one gammarid survived. This gammarid was weak and occasionally showed locomotion. In contrast, two niphargids survived the copper pulse and the subsequent recovery period. The two niphargids were more active during the recovery phase and were stronger when the experiment was stopped. The locomotion patterns of the two species during the recovery period were very similar but both niphargids were way
D. magna had lower LC50 values of 58 μg/l after exposure and 21 μg/l after seven days recovery. Unfortunately, mortality of the control group after recovery was ~50%, implying poor health of individuals or handling stress. Since both species’ LC50 values in literature are strongly overlapping, a clear decision regarding their sensitivity cannot be drawn. A study trying to compare their sensitivity to a variety of substances came to the result of similar sensitivity [4, 44].
During the laboratory experiments the two tested stygal species appeared to be more tolerant to acute copper pulses, although long lasting exposure had strong effect on P. slavus. 24- hour copper pulses showed no effect on either species’ mortality. During the recovery period, there was likewise no change in survival rate. Their high tolerance may be due to their adaptation to groundwater conditions. In groundwater, the most limiting parameters appear to be food and oxygen supply . Therefore, groundwater species possess a slower metabolism, lower basal activity and a longer life expectation compared to their relatives in surface waters. Slower metabolism and adaptation to low oxygen levels may be advantageous during copper exposure, since toxicokinetics act slower and gill impairment may not have as severe effects. Intriguingly regarding the behavioural responses, N. casparyi showed very similar behavioural changes to copper pollution as G. fossarum during the laboratory pulse experiments and the stepwise increasing copper pulse.
Besides the fixed endpoint evaluation using LC50, we also determined the time to reach 50% effect [ET50]. Mean ET50 values towards 500 μg/l copper for G.fossarum, N.casparyi, and D.magna are 9.9h, 6.5h, and 5.8 hours, respectively. The additional data for G. fossarum and D. magna match with the previously determined sensitivity using mortality as parameter. Daphnids’ activity reduced by 50% 4.1 hours earlier than gammarids did. In this regard, faster response argues for higher sensitivity of daphnids towards copper. Surprisingly, N. casparyi revealed strong behavioural changes. Reduction of locomotion by 50% indicates impact on the organisms. They reduced locomotion 0.7 hours later than daphnids, but 3.4 hours earlier than gammarids. Here N. casparyi appear to be likewise more sensitive than G. fossarum or alternatively respond more pronounced. Strong reduction in activity and short reaction time of N. casparyi indicate behavioural sensitivity, despite higher tolerance according to their higher survival rate. Using the ET50 approach we were able to assess and compare N. casparyis’ sensitivity, although they did not show increased mortality after exposure. Thus, this approach not only gave us the opportunity to include N. casparyi into the comparison, it furthermore provides higher resolution in cases of ties and thus may in future reduce the number of individuals needed to distinguish sensitivity. Unfortunately, in contrast to fixed endpoint evaluation [LC50], time-to-effect determination is only sparsely used to assess sensitivity towards pollutants, despite its advantages.
Eucyclops serrulatus, a test species of a so far unpublished study, appear to be the most sensitive species towards copper pollution in our acute toxicity tests. 24-h LC50 was determined as being 25 μg/l. LC50 further decreased with increasing exposure time for 48-h, 72-h, 96-h, and 7-d to 19 μg/l, 17 μg/l, 17 μg/l, and 16 μg/l, respectively [Gerhardt, in preparation]. Even low concentrations of 25μg/l resulted in death of 50% of the individuals within 24 hours. Furthermore, in several laboratory copper pulse experiments using LimCo`s Microimpedance Sensor System [MSS] for small microscopic organisms, E. serrulatus showed promising, reproducible behavioural changes [Gerhardt, in preparation].
The difference in acute and chronic toxicity may be due to dependence on species specific bioavailability, the rate of uptake, Cu homeostasis mechanisms, and furthermore depend on physicochemical conditions . According to the congruent literature data copper toxicity increases with increasing temperature, decreasing DOC, pH, major cations and hardness [17, 18, 62]. Furthermore, LC50 for all species decreases with exposure time.
Beyond the implication for the directly affected organisms through acute or chronic exposure, this has also consequences for other members of the community and thus the whole ecosystem, since the presented species represent important species of the lower trophic levels report about the top-down control of D. magna over algal blooms [15, 16, 65]. Furthermore, diminishing low trophic levels also leads to strong bottom-up effects [22, 77] decreasing predator abundance i.e. fishes and therefore affecting fisheries . Beside these more prominent effects of surface water bodies, disturbances of water communities have huge impact on water body services. For groundwater these were reviewed by Griebler . but because there is high interaction between surface and groundwater, they strongly depend on each other . Groundwater ecosystem services include
“1 purification of water and its storage in good quality for decades and centuries.
2 active biodegradation of anthropogenic contaminants and inactivation and elimination of pathogens.
3 nutrient recycling, and
4 mitigation of floods and droughts.”
Accordingly, species richness, abundance and activity are crucial for maintaining these services.
During our second copper pulse experiment using G. fossarum and D. magna, a similar reaction could be observed, although the reaction was not as pronounced in neither of the employed species. The reduction in variability combined with constant high activity was a first and almost immediate alarm sign. This early response was followed by a delayed and strong reduction in activity of G. fossarum and D. magna in the MFB. They reduced their activity by >50% after 8.7 and 6.6 hours, respectively, representing their ET50. The time to effect determination allows for another adjustable parameter for monitoring. The two species in the MFB reduced their activity almost alike. Differences in ET50 are mainly due to a single drop in activity that crossed EC50 of D. magna but closely missed EC50 of G. fossarum. As a last irreversible effect, one or multiple animals showing no activity can be determined as immobile/dead.
During the stepwise increasing copper pulse, the BD TI only started to increase upon 500 μg/l copper. This was 4.5 hours later than G.fossarum and N. casparyi. During the second pulse experiment, BD TI rose 1.5 hours after exposure. Compared to the initial response of the MFB species, response is late but still earlier than the subsequent severe decrease in activity. The BD TI is an integration of multiple behavioural parameters like average speed, swimming height, distance, and others. Based on these factors anomalies lead to elevation of the BD TI. Heightened BD TI values before start of the 500 μg/l pulse may have already previously generated an alarm signal and indicate problematic preconditions like wrong number of daphnids, poor health, stress or dead daphnids. In contrast to the MFB, the BD heats the incoming water to 20°C, artificially illuminate the test chamber and contain algae as food source. Heating and filtering the water, both additional efforts, portray “interference” in the natural conditions. Higher water temperature increases metabolism and might cause a rise in “sensitivity due to higher metabolism”. This effect may not reflect natural conditions, therefore seem unsuitable for field experiments and furthermore hampers extrapolation of laboratory experimental results into implication on ecosystems . The same is true for artificial light. Although the light source probably does not produce UV light, light may result in a positive or negative compound toxicity . Furthermore pollutants were shown to alter circadian rhythms in the field, implications that would be hidden if artificial light was implemented permanently . During the field experiment, in each period, the BD was maintained several times and daphnids were exchanged at least twice. Together with the two cultivations [algae and daphnids], this highlight higher maintenance efforts compared to the MFB.
Further online biomonitors base on luminescence of bacteria, growth or photosynthesis of algae or behavioural changes of multicellular species like crustacean or fish [5, 19, 71]. Biological early warning systems [BEWS] have to fulfil several properties: The system must be affordable in acquisition and maintenance, it must respond fast with high accuracy, it should have a broad applicability, and the selected test organism must be sensitive and appropriate for the demands of application and the prevalent conditions. Despite the substantial progress made in recent years, the authors highlight the lack of a device comprising all desired characteristics. Microsensor and nanotechnology based systems appear too expensive and often not reliable. Devices based on algae or other species’ growth show an unfavourable lag time in response . Systems using aquatic animals are usually restricted in the deployment of a particular species which confines the application field due to their sensitivity. Furthermore, depending on the species, a second culture [algae] may be needed as food for daphnids, which in turn increases maintenance efforts.
During the present study the MFB proved to be affordable in acquisition and low in maintenance efforts due to its simple and robust technology, setup and handling. Second, since the MFB is not restricted to a particular species, we successfully used both surface and groundwater crustaceans as well as microscopic species such as D. magna and E. serrulatus. Because the MFB is not based on optical measurements, a variety of different species and media [unfiltered water, sediment] can be applied, which enable the application to a wide range of areas [32, 33, 39, 42, 70]. The MFB can operate with untreated raw water [no filtration, no heating needed] in the waterworks, hence reduces the maintenance efforts and recording of artefacts, and increases the ecological relevance of monitoring data. Because of this, the realistic in situ availability and abiotic conditions of the chemicals are not changed and locally resident species can be used as bioindicator species in the biomonitor. Tests using G. fossarum or N. casparyi showed that maintenance intervals of >14 days in the MFB are conceivable. These are larger than the usual stand-alone time for biomonitors using daphnids, which is on week.
Experiments using copper as toxicant showed toxic effect of chronic exposure on P. slavus, but not in acute pulses, excluding it from monitoring usage. Our study revealed fast, sensitive responses in N. casparyi similar to G. fossarum. However, difficult capture and yet unknown breeding conditions represent high hurdles to use these groundwater species in continuous biomonitoring of groundwater and drinking water. Furthermore, the lack of toxicity data for stygal species display a problem regarding the determination of applicability.
D. magna proved its applicability as biomonitoring species in several studies. Nevertheless, providing a piece of an alder leave as food source for G. fossarum is feasible and very low in effort. Thus, maintenance cost and time is low. High abundance and broad distribution in water bodies qualify G. fossarum as a common indicator for pollution. The large availability of toxicity data towards numerous natural or anthropogenic compounds enable the use of G. fossarum in a broad range of applications. Furthermore, it represents a robust, but still sensitive and fast reacting species. Therefore, we recommend G. fossarum as a suitable species for biomonitoring in a wide range of fields.
Furthermore, field experiments showed escape responses of G. fossarum and N. casparyi, that are faster than the established biomonitor using daphnids. Throughout the time in the waterworks, the MFB represented a valuable tool for drinking water intake surveillance. Through its high flexibility, the MFB portrays a useful device for future water quality control in a wide range of fields.
This project is supported by the German Federal Ministry of Education and Research [BMBF] as part of the funding measure “Regional Water Resources Management for Sustainable Protection of Waters in Germany” – ReWaM [GroundCare: funding code 033W037H]
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